QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.116954v1 321/1/98    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Huang, C.-W. Articles by Wu, S.-N. Search for Related Content PubMed PubMed Citation Articles by Huang, C.-W. Articles by Wu, S.-N.

NEUROPHARMACOLOGY

Activation by Zonisamide, a Newer Antiepileptic Drug, of Large-Conductance Calcium-Activated Potassium Channel in Differentiated Hippocampal Neuron-Derived H19-7 Cells

Department of Neurology (C.-W.H.) and Institute of Clinical Medicine (C.-W.H., C.-C.H.), Department of Pediatrics (C.-C.H.), and Department of Physiology and Institute of Basic Medical Sciences (S.-N.W.), National Cheng Kung University Medical College, Tainan, Taiwan

Received November 15, 2006; accepted January 23, 2007.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Zonisamide (ZNS; 3-sulfamoylmethyl-1,2-benzisoxazole), as one of the newer antiepileptic drugs, has been demonstrated its broad-spectrum clinical efficacy on various neuropsychiatric disorders. However, little is known regarding the mechanism of ZNS actions on ion currents in neurons. We thus investigated its effect on ion currents in differentiated hippocampal 19-7 cells. In whole-cell configuration of patch-clamp technology, the ZNS (30 µM) reversibly increased the amplitude of K⁺ outward currents, and paxilline (1 µM) was effective in suppressing the ZNS-induced increase of K⁺ outward currents. In inside-out configuration, ZNS (30 µM) applied to the intracellular face of the membrane did not alter single-channel conductance; however, it did enhance the activity of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels primarily by decreasing mean closed time. In addition, the EC₅₀ value for ZNS-stimulated BK_Ca channels was 34 µM. This drug caused a left shift in the activation curve of BK_Ca channels, with no change in the gating charge of these channels. Moreover, ZNS at a concentration greater than 100 µM also reduced the amplitude of A-type K⁺ current in these cells. A simulation modeling based on hippocampal CA3 pyramidal neurons (Pinsky-Rinzel model) was also analyzed to investigate the inhibitory effect of ZNS on the firing of simulated action potentials. Taken together, this study suggests that, in hippocampal neurons during the exposure to ZNS, the ZNS-mediated effects on BK_Ca channels and A-type K⁺ current could be potential mechanisms through which it affects neuronal excitability.

The large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels, the products of a nearly ubiquitous, alternatively spliced gene (SLO or KCNMA1) (Butler et al., 1993), are distinguished from other K⁺ channels in that they are gated open in response to Ca²⁺ binding to sites located inside of the channel, as well as by depolarized membrane potentials (Ghatta et al., 2006; Wu et al., 2006). Their activities provide a link between the metabolic and electrical state of cells because they are sensitive to stimulation by increased intracellular Ca²⁺. BK_Ca channels in hippocampal pyramidal cells have been identified for their collaborative effect with voltage-gated K channels in repolarizing spike and fast after hyperpolarization (Storm, 1990; Shao et al., 1999). In addition, riluzole and cilostazol have been reported to enhance the activity of BK_Ca channels in pituitary tumor (GH₃) cells (Wu and Li, 1999; Wu et al., 2004).

A recent study has demonstrated that mutation of the pore-forming -subunit of BK_Ca channels could cause generalized epilepsy, paroxysmal dyskinesia (Du et al., 2005), and mental deficiency disorder such as autism (Laumonnier et al., 2006). In addition, the increased activity of BK_Ca channels, possibly associated with the peripheral antinociceptive action of metamizol and meloxicam (Ortiz et al., 2003, 2005), could counteract the deleterious effects of excitatory neurotransmitters following neurotoxic or ischemic injuries as well (Gribkoff et al., 2001; Ghatta et al., 2006). However, it remains unknown whether ZNS, as a potential neuropsychiatric modulator, produces any effects on the activity of these channels.

The hippocampal neuron-derived H19-7 cell line is known to possess the characteristics of hippocampal neurons (Morrione et al., 2000; Bhargava et al., 2002). This cell line has been used for studies on the development, plasticity, and commitment in hippocampal neurons (Morrione et al., 2000; Bhargava et al., 2002). Therefore, the goals of this study were to examine the effect of ZNS on K⁺ outward current in differentiated hippocampal H19-7 cells, to address the issue of whether this drug can affect the activity and kinetic properties of BK_Ca channels, and to determine whether it affects other types of ion currents present in these cells. Interestingly, these results indicate that ZNS can directly enhance the activity of BK_Ca channels.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Cell Preparation. The H19-7 cell line was originally derived from hippocampi dissected from embryonic day 17 Holtzman rat embryos and immortalized by retroviral transduction of temperature-sensitive tsA58 SV40 large T antigen. This cell line was obtained from American Type Culture Collection ([CRL-2526]; Manassas, VA) (Morrione et al., 2000; Huang et al., 2004). Cells were maintained in Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/l sodium bicarbonate and 4.5 g/l glucose supplemented with 10% fetal bovine serum, 200 µg/ml G418, and 1 µg/ml puromycin in flasks that were coated with 15 µg/ml poly-L-lysine. They were equilibrated in a humidified atmosphere of 5% CO₂/95% at an air temperature with a permissive temperature of 34°C. The experiments were generally performed after 5 days of subcultivation (60–80% confluence) with cells obtained from passages 2 and 4.

Cell Differentiation. For differentiation experiments, hippocampal H19-7 cells were plated at a density of 10⁵ cells/35-mm plate in a medium containing serum at 34°C. After 18 h, the cells were washed extensively and then shifted to a nonpermissive temperature (39°C) in N2 serum-free medium (i.e., Dulbecco's modified Eagle's medium-high glucose medium supplemented with 0.11 mg/ml sodium pyruvate, 2 mM glutamine, 0.1 mg/ml transferrin, 20 nM progesterone, 0.1 mM putrescine, and 30 nM sodium selenite), supplemented with 50 ng/ml insulin-like growth factor-I (Life Technologies, Grand Island, NY). After 48 h, the cells were harvested and observed for neurite formation and subsequent electrophysiological experiments (Kim et al., 1997; Morrione et al., 2000). Therefore, rat hippocampal neurons-derived H19-7 cells could either proliferate at the permissive temperature of 34°C in response to insulin-like growth factor or differentiate to a neuronal phenotype in N2 medium at the nonpermissive temperature of 39°C (Kim et al., 1997; Morrione et al., 2000; Huang et al., 2006).

Electrophysiological Measurements. Immediately before each experiment, differentiated H19-7 cells were dissociated, and an aliquot of cell suspension was transferred to a recording chamber positioned on the stage of an inverted microscope (DM IL; Leica Microsystems, Wetzlar, Germany). Cells were bathed at room temperature (20–25°C) in normal Tyrode's solution containing 1.8 mM CaCl₂. The recording pipettes were pulled from Kimax-51 glass capillaries (Kimble Glass, Vineland, NJ) using a two-stage microelectrode puller (PP-830; Narishige, Tokyo, Japan), and the tips were fire-polished with a microforge (MF-83; Narishige). When filled with pipette solution, their resistance ranged between 3 and 5 M. Ion currents were recorded in a whole-cell or inside-out configuration of the patch-clamp technique, using an RK-400 patch-clamp amplifier (Bio-Logic, Claix, France) (Huang et al., 2006). All potentials were corrected for liquid junction potential, a value that would always develop at the tip of the pipette when the composition of the pipette solution was different from that in the bath. Tested drugs were applied through perfusion or added to the bath to obtain the final concentration indicated.

Data Recordings and Analyses. The signals consisting of voltage and current tracings were displayed on an analog/digital oscilloscope (HM 507; Hameg Inc., East Meadow, NY). The data were stored online in a laptop computer through an analog/digital interface (Digidata 1322A; Molecular Devices, Union City, CA), controlled by a commercially available software package (pCLAMP 9.0; Molecular Devices). The sampling rate for electrophysiological measurements was 10 kHz. Currents were low-pass filtered at 1 or 3 kHz. Ion currents recorded during experiments were stored and analyzed subsequently using pCLAMP 9.0 software (Molecular Devices), Origin 7.5 software (Microcal Software, Inc., Northampton, MA), SigmaPlot 7.0 software (SPSS, Inc., Apex, NC), or custom-made macros in Excel 2003 (Microsoft, Redmond, WA). The pCLAMP-generated voltage-step protocols were generally used to examine the current-voltage (I-V) relations for ion currents.

To calculate concentration-dependent stimulation of ZNS on the activity of BK_Ca channels, H19-7 cells were bathed in a high-K⁺ (145 mM) solution, and inside-out configuration was performed. Each excised patch was held at +60 mV, and the bath medium contained 0.1 µM Ca²⁺. The concentration-dependent effect of ZNS at different concentrations (3–100 µM) was fitted with a Hill function. That is, relative open probability = (E_max x [C]^nH)/(EC₅₀^nH + [C]^nH), where [C] represents the ZNS concentration, n_H and EC₅₀ are Hill coefficients and the ZNS concentration needed to increase channel activity by 50%, respectively; and E_max is the ZNS-induced maximal stimulation of BK_Ca channels.

Single-channel currents of BK_Ca or K_ATP channels were analyzed using pCLAMP 9.0 software (Molecular Devices). Multigaussian adjustments of the amplitude distributions between channels were used to determine unitary currents. The functional independence between channels was verified by comparing the observed stationary probabilities with the values calculated according to binomial law. The number of active channels in the patch N was counted at the end of each experiment and then used to normalize opening probability at each potential. The open probabilities were evaluated using an iterative process to minimize the ² calculated with a sufficiently large number of independent observations. The open or closed distribution of the channel was fit with a one- or two-exponential function.

The plots of the open probability of BK_Ca channels as a function of membrane potential were constructed and fit with a Boltzmann function using nonlinear regression analysis with the Origin 7.5 (Microcal). That is, relative open probability = n_p/{1 + exp[– (V – V_1/2)/k]}, where n_p = the maximal probability of channel openings, V = the membrane potential in mV, V_1/2 = the membrane potential for half-maximal activation, and k = the slope factor of the activation curve.

The averaged results are presented as the mean values ± S.E.M. The paired or unpaired Student's t test and one-way analysis of variance with the least significance difference method for multiple comparisons were used for statistical evaluation of the differences among the mean values. Differences between values were considered significant when p < 0.05 or <0.01.

Drugs and Solutions. Zonisamide (Zonegran) was kindly provided by Dainippon Pharmaceuticals (Osaka, Japan). Riluzole was obtained from Tocris Cookson Ltd. (Bristol, UK). Tetraethylammonium chloride and tetrodotoxin were purchased from Sigma Chemical (St. Louis, MO), apamin and iberiotoxin were from Alomone Laboratories (Jerusalem, Israel), and paxilline was from Biomol (Plymouth Meeting, PA) (Wu et al., 2006). All other chemicals were commercially available and of reagent grade. The twice-distilled water that had been deionized through a Millipore-Q system (Millipore Corporation, Billerica, MA) was used in all experiments.

The composition of normal Tyrode's solution was 136.5 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.53 mM MgCl₂, 5.5 mM glucose, and 5.5 mM HEPES-NaOH buffer, pH 7.4. To record K⁺ currents, a patch pipette was filled with a solution consisting of 140 mM KCl, 1 mM MgCl₂, 3 mM Na₂ATP, 0.1 mM Na₂GTP, 0.1 mM EGTA, and 5 mM HEPES-KOH buffer, pH 7.2. To record Ca²⁺ current, KCl inside the pipette solution was replaced with equimolar CsCl, and pH was adjusted to 7.2 with CsOH. In single-channel recordings, high-K⁺ bathing solution contained 145 mM KCl, 0.53 mM MgCl₂, and 5 mM HEPES-KOH, pH 7.4, and the pipette solution contained 145 mM KCl, 2 mM MgCl₂, and 5 mM HEPES-KOH, pH 7.2.

Mathematical Modeling. Spontaneous discharge of neurons was simulated using the model of hippocampal CA3 pyramidal neurons, which was originally derived from Pinsky and Rinzel (1994). The important role of CA3 neurons in kindling and temporal lobe seizures has been established (Jang et al., 2006). This model consists of a fast and transient Na⁺ current, a persistent, depolarization-activated Na⁺ current, a low-threshold Ca²⁺ current, a high-threshold Ca²⁺ current, a Ca²⁺-activated K⁺ current, a transient outward K⁺ current, a slowly inactivating K⁺ current, and a hyperpolarization-activated cation current. The source file for this model is readily available at http://cams.njit.edu/~vbooth/. The solutions to the different sets of ordinary differential equations in the simulations were approximated using the X-Win32 version of XPPAUT on a Dell Precision 670 workstation (Round Rock, TX) (Ermentrout, 2002; Wu and Chang, 2006). The conductance values and reversal potentials used to solve the set of differential equations are lised in Table 1.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Stimulatory Effect of ZNS on K⁺ Outward Currents in Differentiated Hippocampal H19-7 Cells. In the initial set of experiments, the whole-cell configuration of the patch-clamp technique was used to investigate the effect of ZNS on macroscopic K⁺ outward current (I_K). When H19-7 cells were bathed in normal Tyrode's solution containing 1.8 mM CaCl₂, I_K in response to a 1-s-long ramp pulse could be elicited. This current was found to be outwardly rectifying. Within 1 min of exposing the cells to ZNS (30 µM), the amplitude of I_K evoked by the ramp pulse was significantly increased (Fig. 1). For example, at the level of +80 mV, ZNS (30 µM) significantly increased the amplitude of I_K from 795 ± 59 to 1509 ± 74 pA (p < 0.05, n = 6). In addition, ZNS (30 µM) increased the slope conductance of I_K at the level of +40 mV from 16.7 ± 2.3 (n = 5) to 24.2 ± 3.5 (n = 5) nS and at the level of +80 mV from 18.4 ± 2.5 to 35.7 ± 3.6 nS (n = 5). After washout of ZNS, the amplitude of I_K returned to 822 ± 31 pA (n = 5). A further application of 1 µM paxilline could reduce I_K to 812 ± 63 pA (p < 0.05, n = 5). In addition, iberiotoxin (200 nM), but not apamin (200 nM), significantly reversed the ZNS-induced increase of I_K. In addition, the value for reversal potential of I_K measured in the presence of ZNS or ZNS plus paxilline was around –75 mV. The results suggest that the amplitude of I_K in response to the ramp pulses can be sensitive to increases in ZNS. The ZNS-induced increase of I_K can be blocked by paxilline or iberiotoxin.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Effect of ZNS on IK in differentiated H19-7 cells. In these experiments, cells were bathed in normal Tyrode's solution containing 1.8 mM CaCl2 and CdCl2 (0.5 mM). Each cell was held at –50 mV, and the ramp pulses from –80 to +100 mV with a duration of 1 s were applied. A, original current traces showing the effect of ZNS on IK obtained with or without a further application of paxilline. ZNS, 30 µM; paxilline (Pax), 1 µM. B, bar graph showing the effect of ZNS on IK in the absence and presence of paxilline, iberiotoxin, or apamin. The amplitude was measured at the level of +80 mV. ZNS, 30 µM; Pax, 1 µM; iberiotoxin (Iber), 200 nM; apamin (Apa), 200 nM. Each point represents the mean ± S.E.M. (n = 4–9). *, p < 0.05 versus control. **, p < 0.05 versus ZNS-alone group.

Stimulatory Effect of ZNS on the Activity of BK_Ca Channels in Differentiated H19-7 Cells. Paxilline and iberiotoxin, known inhibitors of BK_Ca channels, could reverse ZNS-stimulated I_K. The increased macroscopic I_K could be due to the increased open probability, an increase in the number of active channels, or both. The effect of ZNS on BK_Ca channel activity in these cells was further investigated (Fig. 2). In these experiments, single-channel recordings with an inside-out configuration were performed in symmetrical K⁺ solution (145 mM) (Fig. 2A). Bath medium contained 0.1 µM Ca²⁺, and holding potential was held at +60 mV. When ZNS (100 µM) was applied to the intracellular surface of the excised patch, the channel activity was significantly increased to 0.015 ± 0.004 (n = 5) from a control of 0.004 ± 0.001 (n = 5, p < 0.05). A further application of paxilline (1 µM) could decrease the channel activity to 0.005 ± 0.001 (n = 5). The channel activity in paxilline (1 µM) alone was 0.003 ± 0.001 (n = 5). Application of ZNS (3–1000 µM) was found to enhance the probability of channel openings in a concentration-dependent manner. The half-maximal concentration required for the stimulatory effect of ZNS on BK_Ca channel activity was 34 µM, and Hill coefficient was 1.2. Thus, the ZNS-induced increase in the amplitude of I_K in these cells could be associated with its increase in the open probability of BK_Ca channels, rather than changes in the number of active channels.

View larger version (32K): [in this window] [in a new window]   Fig. 2. Stimulatory effect of ZNS on BKCa channel activity in differentiated H19-7 cells. A, each excised patch was held at +60 mV. Channel activity during the exposure to ZNS (1 mM) was taken to be 1.0, and those at different concentrations of ZNS were then compared. B, relationship between the ZNS concentration and BKCa channel activity. Red smooth line, best fit to the Hill equation. The values for EC50 and the Hill coefficient were 34 and 1.2 µM, respectively. C, bar graph showing the effect of ZNS, ZNS with addition of Pax, and Pax alone on IK. Each point represents the mean ± S.E.M. (n = 5–10). *, p < 0.05 versus control. **, p < 0.05 versus ZNS-alone group. Pax, 1 µM.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Effect of ZNS on the I-V relationship and the Ca2+ sensitivity of BKCa channels in differentiated H19-7 cells. Experiments in excised patches were conducted in symmetrical K+ solution (145 mM) and a bath medium containing 0.1 µM Ca2+. A, averaged I-V relationship of BKCa channels in the absence () and presence () of 100 µM ZNS. Data are the mean ± S.E.M. (n = 6–10). Notably, there is no significant change in single-channel conductance between the absence and presence of ZNS. B, relationships between membrane potential and the open probability of BKCa channels obtained in the absence () and presence () of 30 µM ZNS. Data are the mean ± S.E.M. (n = 5–8). C, lack of ZNS effect on Ca2+ sensitivity of BKCa channels. Potential was held at +60 mV, and various concentrations of Ca2+ in the bath were applied before and during exposure to ZNS (30 µM). Data are the mean ± S.E.M. (n = 4–6). *, significantly different from controls (i.e., in the absence of ZNS) (p < 0.05).

Effect of ZNS on the Activation Curve of BK_Ca Channels. Figure 3B shows the activation curve of BK_Ca channels in the absence and presence of ZNS (30 µM). The plot of open probability of channel openings as a function of membrane potential was fitted with a Boltzmann function as described under Materials and Methods. In control, n_p = 1, V_1/2 = 56 ± 5 mV, and k = 11.6 ± 2.1 mV (n = 6), whereas in the presence of ZNS (100 µM), n_p = 1.6 ± 0.2, V_1/2 = 42 ± 6 mV, and k = 11.5 ± 1.9 mV (n = 6). The data thus indicate that the ZNS-increased channel activity can be accompanied by a significant shift of the steady-state activation toward less positive potentials, although the slope factor of activation curve remained unaltered. Taken together, ZNS applied to the intracellular surface of the channel is capable of affecting the activation curve with no change in the gating charge of these channels.

Effect of Internal Ca²⁺ Concentrations on ZNS-Stimulated BK_Ca Channel Activity. We next examined whether the ZNS-induced increase in the activity of these channels is associated with the level of internal Ca²⁺ concentrations. In these experiments, when an excised patch was formed, various concentrations of Ca²⁺ in the bath before and during exposure to ZNS (30 µM) were applied. As shown in Fig. 3C, the extent of ZNS-stimulated BK_Ca channels was not altered by changes in the level of intracellular Ca²⁺ concentration. For example, at the level of +60 mV, ZNS (30 µM) increased the open probability at an internal Ca²⁺ concentration of 0.1, 1, and 10 µM to a similar magnitude (i.e., 1.6-fold).

Effect of ZNS on Kinetic Behavior of BK_Ca Channels in Differentiated H19-7 Cells. The effect of ZNS on mean open and closed time of BK_Ca channels in these cells was examined and analyzed during recordings from patches showing only single-channel openings. As shown in Fig. 4, in control cells, the closed time histogram of BK_Ca channels at +60 mV can be fitted by a two-exponential function with a mean closed time of 5.2 ± 0.9 and 54.5 ± 1.6 ms (n = 5). ZNS (30 µM) decreased the lifetime of the closed time to 3.2 ± 0.8 and 19.6 ± 1.1 ms (n = 5, p < 0.05). However, no change in the component of open time distribution was observed in the presence of ZNS (30 µM) (1.62 ± 0.05 versus 1.64 ± 0.04 ms, n = 5, p > 0.05). Taken together, the data suggest that the stimulation by this drug of BK_Ca channels in these cells is largely due to an increase in open probability accompanied by the decreased closed time.

View larger version (30K): [in this window] [in a new window]   Fig. 4. Effect of ZNS on mean open (top) and closed (bottom) time of BKCa channels in differentiated H19-7 cells. Inside-out configuration was performed in these experiments, and the potential was held at +60 mV. Cells were bathed in symmetrical K+ solution (145 mM), and bath medium contained 0.1 µM Ca2+. The open (top) and closed (bottom) time histograms after application of ZNS are shown in the right. Control data were obtained from measuring 296 channel openings with a total record time of 1 min, whereas those obtained in the presence of ZNS were measured from 339 channel openings with a total record time of 30 s. The dashed lines shown in each lifetime distribution are placed at the values of the time constant in an open or closed state.

Effect of Riluzole on BK_Ca Channels in Differentiated Hippocampal H19-7 Cells. Riluzole has been previously reported as stimulating BK_Ca channels (Wu and Li, 1999). We also examined whether the stimulatory effects of ZNS and riluzole on these channels are additive. Interestingly, as shown in Fig. 5, riluzole (30 µM) increased the channel open probability; however, a subsequent application of ZNS (30 µM) did not increase the channel activity further. Riluzole (30 µM) significantly increased the probability of channel openings from 0.12 ± 0.02 to 0.24 ± 0.03 (n = 6, p < 0.05). There was no significant difference in channel activity between the presence of riluzole alone and ZNS plus riluzole [0.24 ± 0.03 (n = 6) versus 0.23 ± 0.03 (n = 6), p > 0.05]. However, application of cilostazol (30 µM) could increase the open probability further when ZNS or riluzole was continuously present in the bath. Cilostazol was reported as an opener of BK_Ca channels (Hong et al., 2003; Wu et al., 2004). Taken together, the results led us to suggest that riluzole potentially occluded the effect of zonisamide, although these two drugs can be effective in enhancing channel activity.

View larger version (47K): [in this window] [in a new window]   Fig. 5. Effects of riluzole, ZNS, riluzole plus ZNS, ZNS plus cilostazol, and riluzole plus cilostazol on the activity of BKCa channels. Channel activity of each excised patch was measured at +60 mV. ZNS, 30 µM zonisamide; Ril, 30 µM riluzole; Cil, 30 µM cilostazol. A, channel activities with ZNS or riluzole plus cilostazol, cilostazol was subsequently applied in the continued presence of ZNS or riluzole. B, bar graphs show the relative open probability in these experiments. Each point represents the mean ± S.E.M. (n = 5–9). *, significantly different from control group. **, significantly different from ZNS-alone group. ***, significantly different from the riluzole-alone group.

Effect of ZNS on Voltage-Dependent L-Type Ca²⁺ Current in Differentiated H19-7 Cells. The question may arise whether a ZNS-induced increase in whole-cell I_K is associated with an increased Ca²⁺ influx through voltage-dependent Ca²⁺ channels. ZNS has been previously reported as blocking Ca²⁺ current (Suzuki et al., 1992). The effect of ZNS on the amplitude of L-type Ca²⁺ current (I_Ca,L) in differentiated H19-7 cells was thus assessed. In these experiments, cells were bathed in normal Tyrode's solution containing 1.8 mM CaCl₂, and the recording pipettes were filled with Cs⁺-containing solution. Cell exposure to ZNS (30 µM) did not affect the peak amplitude of I_Ca,L. However, ZNS at a concentration of 300 µM resulted in a decrease in I_Ca,L (Fig. 6). For example, when cells were depolarized from –50 to 0 mV, the peak amplitude of I_Ca,L was slightly but significantly decreased from 85 ± 12 to 79 ± 11 pA (n = 5, p < 0.05). In addition, a further application of nifedipine (0.3 µM), a known blocker of I_Ca,L, almost abolished the amplitude of I_Ca,L. Thus, the results indicate that this drug can stimulate Ca²⁺-activated K⁺ current in a manner unlikely to be associated with an increase in the amplitude of I_Ca,L.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Inhibitory effect of ZNS on voltage-dependent L-type Ca2+ current (ICa,L) in differentiated H19-7 cells. In these experiments, the cells were bathed in normal Tyrode's solution that contained 1.8 mM CaCl2, 1 µM tetrodotoxin, and 10 mM tetraethylammonium chloride. The recording pipettes were filled with a Cs+-containing solution. A, original currents showing the effects of ZNS and ZNS plus nifedipine on ICa,L. The cell was depolarized from –50 to 0 mV at a rate of 0.05 Hz. B, bar graph showing the effect of ZNS on the amplitude of ICa,L with or without addition of nifedipine. ZNS, 300 µM; nifedipine (Nif), 0.3 µM. *, significantly different from control (p < 0.05). **, significantly different from the ZNS-alone group (p < 0.05).

View larger version (21K): [in this window] [in a new window]   Fig. 7. Inhibitory effect of ZNS on IA in differentiated H19-7 cells. Cells were bathed in Ca2+-free solution containing tetrodotoxin (1 µM) and CdCl2 (0.5 mM). A, original current traces showing the effect of ZNS (100 and 300 µM) on IA in these cells. The cells were depolarized from –50 to +50 mV with a duration of 300 ms. B, averaged I-V relationships of IA in the absence () and presence () of ZNS. Each point represents the mean ± S.E.M. (n = 5–7).

Effects of ZNS on Repetitive Firing of Action Potentials in Modeled Hippocampal CA3 Pyramidal Neurons. To determine how ZNS alters the discharge pattern of hippocampal neurons and to address questions that are difficult to be experimentally studied, a simulation model, originally derived from Pinsky and Rinzel (1994), was also implemented. ZNS has been previously reported to block voltage-dependent Na⁺ current (I_Na) (Schauf, 1987; Leppik, 2004). In this modeled neuron, when the I_Na conductance was decreased from 18 to 17 nS, the peak amplitude of I_Na was readily reduced, along with the decreased firing of action potentials (Fig. 8A). When the conductance of Ca²⁺-activated K⁺ current was increased from 20 to 40 nS, the frequency of action potential firing was further reduced (Fig. 8B). As a result, the reduced I_Na and the increased Ca²⁺-activated K⁺ current, which mimics the ZNS action, can combine to cause a reduction in the firing of neuronal action potentials. The simulation results can be used to predict that the activity of BK_Ca channels could contribute to the electrical behavior of hippocampal neurons.

View larger version (29K): [in this window] [in a new window]   Fig. 8. Simulation modeling used to mimic ZNS effects on repetitive firing of action potentials in hippocampal neurons. The model was developed based on the electrophysiological properties of hippocampal CA3 pyramidal neurons as described under Materials and Methods. In A, when the INa conductance was arbitrarily decreased from 18 to 17 nS, the amplitude of INa was readily reduced, together with the decrease in the firing of neuronal action potentials. In B, when the conductance of Ca2+-activated K+ current was increased from 20 to 40 nS in the continued presence of reduced INa conductance, which resembles the effects of ZNS, the action potential firing frequency was further reduced, together with the increased amplitude of Ca2+-activated K+ current in this modeled neuron. The horizontal bar shown in each panel indicates the reduction of INa conductance in combination with or without increased conductance of Ca2+-activated K+ current.

	Discussion

Top Abstract Materials and Methods Results Discussion References

The concentration-dependent stimulation of BK_Ca channel activity with an EC₅₀ value of 34 µM was observed in the presence of ZNS. This concentration of ZNS is compatible with clinically relevant concentrations, ranging from 50 to 200 µM (Peters and Sorkin, 1993; Tasaki et al., 1995; Oommen and Mathews, 1999; Huang et al., 2005). The observed effect of ZNS in this study is likely to occur at a concentration achievable in humans. It is thus reasonable to assume that BK_Ca channels present in neurons are a relevant "target" for the action of ZNS. The ability of ZNS to activate BK_Ca channels may constitute an important component regulating neuronal function, aiding in understanding the broad-spectrum efficacy in epilepsy and various neuropsychiatric disorders.

Our study demonstrates that ZNS does not modify single-channel conductance of BK_Ca channels, although it increases the channel open probability significantly. The increased amplitude of macroscopic I_K is thought to be primarily due to the increased probability of channel openings accompanied by a decrease in mean closed time. In addition, when applied to the internal face of the membrane, it did not merely increase the activity of BK_Ca channels in the patch but also increased a shift of voltage sensitivity of the channel to less positive potentials. The sensitivity to this drug in neurons would be thus expected to depend on the preexisting level of resting potential, the firing rate of action potentials, or the ZNS concentration used.

It is noteworthy that, unlike the molecule of cilostazol, ZNS was found to possess an intramolecular hydrogen bond with a short interatomic distance and an aromatic ring poor of electrons, the properties of which are shared by other inhibitors of carbonic anhydrase (Tricarico et al., 2004). The present results also demonstrate that the stimulatory effect of ZNS and riluzole on the activity of BK_Ca channels is not additive. Riluzole has been previously reported as an opener of BK_Ca channels in pituitary tumor (GH₃) cells (Wu and Li, 1999). It is likely that these two components, which are structurally related, may interact with the same binding site in the channel.

Previous reports have shown that some of the indole diterpenes, which are potent inhibitors of BK_Ca channels, are associated with their tremorogenic activity (Knaus et al., 1994; Smith et al., 1997). Recent reports have demonstrated that compared with blocker, zonisamide is effective in treating essential tremor (Morita et al., 2005; Ondo, 2006). Further studies are needed to determine the extent to which ZNS-stimulated activity of BK_Ca channels contribute to its treatment of essential tremor.

ZNS at a concentration greater than 100 µM was found to block I_A in differentiated hippocampal H19-7 neurons. Lack of A-type K⁺ channels has been proposed to decrease the seizure threshold in the cortical malformation of methylazoxymethanol-exposed rats (Castro et al., 2001). Although no report on ZNS-worsening seizures has been noted, ZNS has been recently reported to improve depressive symptoms in bipolar disorders (Ghaemi et al., 2006). Whether its inhibition of the I_A channels has potential psychotropic benefits in treating underlying cellular disturbances in bipolar disorders, as described previously, remains to be further clarified.

Our simulation results also imply that both blockade of I_Na and direct stimulation of BK_Ca channels caused by ZNS may synergistically act to affect the functional activity of hippocampal neurons in vivo. Taken together, in addition to the blockade of I_Na, our results lead us to suggest that ZNS-mediated antiepileptic action could be partly associated with the direct stimulation of BK_Ca channels expressed in hippocampal neurons.

	Acknowledgements

 
We thank Okada Motohiro (Hirosaki University, Japan) for comments on zonisamide pharmacology. We also thank Ming-Wei Lin for technical assistance.

	Footnotes

 
This work was supported by the National Science Council, Taiwan (Grants NSC-94-2320B-006-019, NSC-94-2314-B-006-040, NSC-95-2314-B-006-046, and NSC-95-2745-B006-002-MY2) and by the Program for Promoting Academic Excellence and Developing World Class Research Centers, Ministry of Education, Taiwan.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.116954.

ABBREVIATIONS: ZNS, zonisamide, 3-sulfamoylmethyl-1,2-benzisoxazole; BK_Ca, large-conductance Ca²⁺-activated K⁺; riluzole, 2-amino-6-trifluoromethoxybenzothiazole; I-V, current-voltage; I_K, K⁺ outward current; I_Ca,L, L-type Ca²⁺ current; I_A, A-type K⁺ current; I_Na, Na⁺ current; Pax, paxilline.

Address correspondence to: Dr. Sheng-Nan Wu, Department of Physiology, National Cheng Kung University Medical College, 1 University Road, Tainan 70101, Taiwan. E-mail: snwu{at}mail.ncku.edu.tw

	References

Top Abstract Materials and Methods Results Discussion References

 

Baulac M (2006) Introduction to zonisamide. Epilepsy Res 68S: S3–S9.

Bhargava A, Mathias RS, McCormick JA, Dallman MF, and Pearce D (2002) Glucorticoids prolong Ca²⁺ transients in hippocampal-derived H19-7 neurons by repressing the plasma membrane Ca²⁺-ATPase-1. Mol Endocrinol 16: 1629–1637.[Abstract/Free Full Text]

Butler A, Tsunoda S, McCobb DP, Wei A, and Salkoff L (1993) mSlo, a complex mouse gene encoding "maxi" calcium-activated potassium channels. Science (Wash DC) 261: 221–224.[Abstract/Free Full Text]

Castro PA, Cooper EC, Lowenstein DH, and Baraban SC (2001) Hippocampal heterotopia lack functional Kv4.2 potassium channels in the methylazoxymethanol model of cortical malformation and epilepsy. J Neurosci 21: 6626–6634.[Abstract/Free Full Text]

Du W, Bautista JF, Yang H, Diez-Sampedro A, You SA, Wang L, Kotagal P, Luders HO, Shi J, Cui J, et al. (2005) Calcium-sensitive potassium channelopathy in human epilepsy and paroxysmal movement disorder. Nat Genet 37: 733–738.[CrossRef][Medline]

Ermentrout GB (2002) Simulating, Analyzing, and Animating Dynamical System: A Guide to XPPAUT for Researchers and Students, Society for Industrial and Applied Mathematics, Philadelphia, PA.

Ghaemi SN, Zablotsky B, Filkowski MM, Dunn RT, Pardo TB, Isenstein E, and Baldassano CF (2006) An open prospective study of zonisamide in acute bipolar depression. J Clin Psychopharmacol 26: 385–388.[CrossRef][Medline]

Ghatta S, Nimmagadda D, Xu XP, and O'Rourke ST (2006) Large-conductance, calcium-activated potassium channels: structural and functional implications. Pharmacol Ther 110: 103–116.[CrossRef][Medline]

Gribkoff VK, Starrett JE Jr, Dworetzky SI, Hewawasam P, Boissard CG, Cook DA, Frantz SW, Heman K, Hibbard JR, Huston K, et al. (2001) Targeting acute ischemic stroke with a calcium-sensitive opener of maxi-K potassium channels. Nat Med 7: 471–477.[CrossRef][Medline]

Hong KW, Kim KY, Shin HK, Lee JH, Choi JM, Kwak YG, Kim CD, Lee WS, and Rhim BY (2003) Cilostazol prevents tumor necrosis factor--induced cell death by suppression of phosphatase and tension homolog deleted from chromosome 10 phosphorylation and activation. J Pharmacol Exp Ther 306: 1182–1190.[Abstract/Free Full Text]

Huang CW, Huang CC, Liu YC, and Wu SN (2004) Inhibitory effect of lamotrigine on A-type potassium current in hippocampal neuron-derived H19-7 cells. Epilepsia 45: 729–736.[CrossRef][Medline]

Huang CW, Huang CC, and Wu SN (2006) The opening effect of pregabalin on ATP-sensitive potassium channels in differentiated hippocampal neuron-derived H19-7 cells. Epilepsia 47: 720–726.[CrossRef][Medline]

Huang CW, Ueno S, Okada M, and Kaneko S (2005) Zonisamide at clinically relevant concentrations inhibits field EPSP but not presynaptic fiber volley in rat frontal cortex. Epilepsy Res 67: 51–60.[CrossRef][Medline]

Jang IS, Nakamura M, Ito Y, and Akaike N (2006) Presynaptic GABAa receptors facilitate spontaneous glutamate release from presynaptic terminals on mechanically dissociated rat CA3 pyramidal neurons. Neuroscience 138: 25–35.[CrossRef][Medline]

Kim B, Leventhal PS, Saltiel AR, and Feldman EL (1997) Insulin-like growth factor-I-mediated neurite outgrowth in vitro requires mitogen-activated protein kinase activation. J Biol Chem 272: 21268–21273.[Abstract/Free Full Text]

Knaus HG, McManus OB, Lee SH, Schmalhofer WA, Garcia-Calvo M, Helms LM, Sanchez M, Giangiacomo K, Reuben JP, and Smith AB 3rd (1994) Tremorogenic indole alkaloids potently inhibit smooth muscle high-conductance calcium-activated potassium channels. Biochemistry 33: 5819–5828.[CrossRef][Medline]

Laumonnier F, Roger S, Guerin P, Molinari F, M'rad R, Cahard D, Belhadj A, Halayem, Persico AM, Elia M, et al. (2006) Association of a functional deficit of the BK_Ca channel, a synaptic regulator of neuronal excitability, with autism and mental retardation. Am J Psychiatry 163: 1622–1629.[Abstract/Free Full Text]

Leppik IE (2004) Zonisamide: chemistry, mechanism of action, and pharmacokinetics. Seizure 13: S5–S9.

Morita S, Miwa H, and Kondo T (2005) Effect of zonisamide on essential tremor: a pilot crossover study in comparison with arotinolol. Parkinsonism Relat Disord 11: 101–103.[Medline]

Morrione A, Romano G, Navarro M, Reiss K, Valentinis B, Dews M, Eves E, Rosner MR, and Baserga R (2000) Insulin-like growth factor I receptor signaling in differentiation of neuronal H19-7 cells. Cancer Res 60: 2263–2272.[Abstract/Free Full Text]

Ondo WG (2006) Essential tremor: treatment options. Curr Treat Options Neurol 8: 256–267.[Medline]

Oommens KJ and Mathews S (1999) Zonisamide: a new anti-epileptic drug. Clin Neuropharmacol 22: 192–200.[Medline]

Ortiz MI, Castañeda-Hernández G, and Granados-Soto V (2003) Possible involvement of potassium channels in peripheral antinociception induced by metamizol: lack of participation of ATP-sensitive K⁺ channels. Pharmacol Biochem Behav 74: 465–470.[CrossRef][Medline]

Ortiz MI, Castañeda-Hernández G, and Granados-Soto V (2005) Pharmacological evidence for the activation of Ca²⁺-activated K⁺ channels by meloxicam in the formalin test. Pharmacol Biochem Behav 81: 725–731.[CrossRef][Medline]

Peters DH and Sorkin EM (1993) Zonisamide: a review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in epilepsy. Drugs 45: 760–787.[Medline]

Pinsky PF and Rinzel J (1994) Intrinsic and network rhythmogenesis in a reduced Traub model for CA3 neurons. J Comput Neurosci 1: 39–60.[CrossRef][Medline]

Rock DM, Macdonald RL, and Taylor CP (1989) Blockade of sustained repetitive action potentials in cultured spinal cord neurons by zonisamide (AD 810, CI 912), a novel anticonvulsant. Epilepsy Res 3: 138–143.[CrossRef][Medline]

Schauf CL (1987) Zonisamide enhances slow sodium inactivation in Myxicola. Brain Res 413: 185–188.[CrossRef][Medline]

Shao LR, Halvorsrud R, Borg-Graham L, and Storm JF (1999) The role of BK-type Ca²⁺-dependent K⁺ channels in spike broadening during repetitive firing in rat hippocampal pyramidal cells. J Physiol (Lond) 521: 135–146.[Abstract/Free Full Text]

Smith BL, McLeay LM, and Embling PP (1997) Effect of the mycotoxins penitrem, paxilline and lolitrem B on the electromyographic activity of skeletal and gastrointestinal smooth muscle of sheep. Res Vet Sci 62: 111–116.[CrossRef][Medline]

Storm JF (1990) Potassium currents in hippocampal pyramidal cells. Prog Brain Res 83: 161–187.[Medline]

Suzuki S, Kawakami K, Nishimura S, Watanabe Y, Yagi K, Seino M, and Miyamoto K (1992) Zonisamide blocks T-type calcium channel in cultured neurons of rat cerebral cortex. Epilepsy Res 12: 21–27.[CrossRef][Medline]

Tasaki K, Minami T, Ieiri I, Ohtsubo K, Hirakawa Y, Ueda K, and Higuchi S (1995) Drug interactions of zonisamide with phenytoin and sodium valproate: serum concentrations and protein binding. Brain Dev 17: 182–185.[CrossRef][Medline]

Tricarico D, Barbieri M, Mele A, Carbonara G, and Camerino DC (2004) Carbonic anhydrase inhibitors are specific openers of skeletal muscle BK channel of K⁺-deficient rats. FASEB J 18: 760–761.[Abstract/Free Full Text]

Wu SN and Chang HD (2006) Diethyl pyrocarbonate, a histidine-modifying agent, directly stimulates activity of ATP-sensitive potassium channels in pituitary GH₃ cells. Biochem Pharmacol 71: 615–623.[CrossRef][Medline]

Wu SN and Li HF (1999) Characterization of riluzole-induced stimulation of large-conductance calcium-activated potassium channels in rat pituitary GH₃ cells. J Investig Med 47: 484–495.[Medline]

Wu SN, Liu SI, and Huang MH (2004) Cilostazol, an inhibitor of type 3 phosphodiesterase, stimulates large-conductance, calcium-activated potassium channels in pituitary GH₃ cells and pheochromocytoma PC12 cells. Endocrinology 145: 1175–1184.[Abstract/Free Full Text]

Wu SN, Wu AZ, and Lin MW (2006) Pharmacological roles of the large-conductance calcium-activated potassium channel. Curr Top Med Chem 6: 1025–1030.[CrossRef][Medline]

Yoshida S, Okada M, Zhu G, and Kaneko S (2005) Effects of zonisamide on neurotransmitter exocytosis associated with ryanodine receptors. Epilepsy Res 67: 153–162.[CrossRef][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.116954v1 321/1/98    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Huang, C.-W. Articles by Wu, S.-N. Search for Related Content PubMed PubMed Citation Articles by Huang, C.-W. Articles by Wu, S.-N.